Cytotoxicity potential of chemical constituents isolated and derivatised from Rhinella marina venom.

Chemical compounds from skin secretions from toads of Bufonidae family have been long-studied. In the search for new molecules with pharmacological action, the 3ß-OH groups of bufadienolides are commonly derivatised using acetyl groups. This work described the isolation and/or structural elucidation of isolated and derivatised compounds from the venom of the Brazilian anuran Rhinella marina, and their evaluation in in vitro assays. In the methanolic extract of the R. marina venom, compound cholesterol (1) was isolated from the CRV-52 fraction by classic column chromatography, dehydrobufotenine (2) by Sephadex LH-20 from the CRV-28 fraction, and a mix of suberoyl arginine (3) and compound 2 was obtained from the CRV-6-33 fraction. The compounds marinobufagin (4), telocionbufagin (5) and bufalin (6) were isolated by classic column chromatography, followed by separation via HPLC in the CRV-70 fraction, and the compound marinobufotoxin (9) was isolated by classic column chromatography in the CRV-6 fraction, here being isolated for the first time in R. marina specimens. Compounds 4 and 5 were submitted for acetylation with acetic anhydride, in the presence of pyridine and 4-dimethyilaminopiridine (DMAP), in order to obtain the compounds 3-acetyl-marinobufagin (7) and 3-acetyl-telocinobufogin (8). The isolated and derivatised compounds were identified by 1H and 13C NMR, and their molecular mass confirmed by mass spectrometry. All compounds (except 1 and 3) were tested in cytotoxic assays by the MTT method and presented cytotoxic potential against human cancer cell lines, as well as against non-tumoral human embryonic kidney HEK-293 cells. With the exception of compound 2, all molecules presented IC50 values < 4 µM, and none caused hemolysis of human erythrocytes, demonstrating a promising cytotoxic potential of natural and chemically-modified bufadienolides. This study presents a detailed contribution of bioactive chemicals from Brazilian Amazon Rhinella species, and indicates promising areas for further studies and pharmaceutical investments.

19-Hydroxy-bufalin, a major bufadienolide isolated from the parotoid gland secretions of the Panamanian endemic toad Rhinella centralis (Bufonidae), inhibits the growth of Trypanosoma cruzi.

American trypanosomiasis is a parasitic neglected disease, responsible for the death of approximately 10,000 people every year. Amphibians are recognized for producing in their cutaneous glands substances with pharmacological potential against a variety of pathologies. Here we investigated the antiprotozoal activity against Trypanosoma cruzi of bufadienolides isolated from the parotoid glands secretions of the toad Rhinella centralis from Panama. NMR and mass spectrometry analysis led to the identification of the active compound 19-hydroxy-bufalin, for which its antitrypanosomal activity and occurrence in the genus Rhinella are reported for the first time. This compound showed low cytotoxicity and significant selectivity which confers to it a potential role for the treatment of Chagas disease.

Cytotoxicity and antimitotic activity of Rhinella schneideri and Rhinella marina venoms.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhinella schneideri and Rhinella marina are toad venoms distributed in different parts of the world, including Brazil, Columbia and amazon. Venoms extracted from different species have many clinical applications such as antimicrobial cardiotonics and treatment of cancer. Aim of the study; In this study, we aim to investigate the effect of venoms extracted from R. schneideri and R. marina on cancer cells and verify possible mechanism of action. MATERIAL AND METHOD: Cytotoxicity analyses was performed using the resazurin reduction assay, where different concentrations of venoms were tested against sensitive CCRF-CEM and P-gp overexpressing ADR/CEM5000 leukemia cells. Programmed cell death was investigated using the flow cytometric annexin V/propidium iodide apoptosis assay. Furthermore, we analyzed flow cytometric cell cycle analyses of CCRF-CEM cells. Effect on tubulin formation was tested using molecular docking and fluorescence microscopy of U2OS-GFP-α-tubulin osteosarcoma cells treated for 24 h with venoms. RESULTS: Cytotoxicity assays revealed a strong activity towards wild-type CCRF-CEM cells (IC50 values of 0.202 ±â€¯0.005 µg/ml and 0.18 ±â€¯0.007 µg/ml for R. schneideri and R. marina, respectively) and multidrug-resistant CEM/ADR5000 cells (IC50 0.403 ±â€¯0.084 µg/ml and 0.32 ±â€¯0.077 µg/ml for R. schneideri and R. marina, respectively). The venoms induced apoptosis as major mechanism of cell death. The venoms induced strong G2/M cell arrest in CCRF-CEM cells. We suggested tubulin as a major target for the venoms. In silico molecular docking of the major constituents of the venoms, i.e. bufalin, marinobufagin, telocinbufagin, hellebrigenin, showed strong binding affinities to tubulin. This result was verified in vitro. The venoms dysregulated microtubule arrangement of U2OS cells expressing GFP-labeled tubulin. Toxicity predictions by QSAR methodology highlighted the toxic features of bufadienolides. CONCLUSION: Our study demonstrated the importance of toad venoms as source of cytotoxic compounds that may serve as lead compounds for the development of novel anticancer drugs.

Toxic activity and protein identification from the parotoid gland secretion of the common toad Bufo bufo.

Anuran toxins released from the skin glands are involved in defence against predators and microorganisms. Secretion from parotoid macroglands of bufonid toads is a rich source of bioactive compounds with the cytotoxic, cardiotoxic and hemolytic activity. Bufadienolides are considered the most toxic components of the toad poison, whereas the protein properties are largely unknown. In the present work, we analysed the cardio-, myo-, and neurotropic activity of extract and the selected proteins from Bufo bufo parotoids in in vitro physiological bioassays carried out on two standard model organisms: beetles and frogs. Our results demonstrate a strong cardioactivity of B. bufo gland extract. The toad poison stimulates (by 16%) the contractility of the insect heart and displays the cardioinhibitory effect on the frog heartbeat frequency (a 27% decrease), coupled with an irreversible cardiac arrest. The gland extract also exhibits significant myotropic properties (a 10% decrease in the muscle contraction force), whereas its neuroactivity remains low (a 4% decrease in the nerve conduction velocity). Among identified peptides present in the B. bufo parotoid extract are serine proteases, muscle creatine kinase, phospholipid hydroperoxide glutathione peroxidase, cytotoxic T-lymphocyte protein, etc. Some proteins contribute to the cardioinhibitory effect. Certain compounds display the paralytic (myo- and neurotropic) properties. As the toad gland extract exhibits a strong cardiotoxic activity, we conclude that the poison is a potent agent capable of slaying a predator. Our results also provide the guides for the use of toad poison-peptides in therapeutics and new drug development.

Mechanism of Rhinella icterica (Spix, 1824) toad poisoning using in vitro neurobiological preparations.

The biological activity of Rhinella icterica toxic secretion (RITS) was evaluated on chick neuromuscular junctions, rat heart́s tissue and mice hippocampal slices. At chick biventer cervicis preparation, RITS (5, 10 and 20µg/mL) produced a concentration-independent irreversible neuromuscular blockade, which was preceded by a transitory increase of muscle twitch tension with the lowest concentration, in 120min recordings. In this set of experiments, RITS incubation partially prevented the curare neuromuscular blockade. The assessment of chick biventer cervicis muscle acetylcholinesterase (AChE) in the presence of RITS showed a significant inhibition of the enzyme, similarly to neostigmine. The incubation of muscles with digoxin or ouabain mimicked the poison activity by increasing the amplitude of the twitches followed by a progressive depression of the muscle strength. In addition, RITS demonstrated a digitalic-like activity, by inhibiting significantly the cardiac Na+, K+-ATPase. When the central nervous system was accessed, RITS induced an increase in the cell viability, in the lowest concentration. In addition, the poison protected slices subject to oxygen/glucose deprivation. Altogether, these data indicate that the poisonous extract of R. icterica is able to interfere with peripheral and central neurotransmission, probably due to a direct interaction with AChE, calcium channels and Na+, K+-ATPase. A further investigation upon the poison toxic components will unveil the components involved in such a pharmacological activity and the potential biotechnological application of this poison.

Antiproliferative activity and chemical composition of the venom from the Amazonian toad Rhinella marina (Anura: Bufonidae).

Little is known on the composition of Peruvian Amazon toad venoms. The large toad Rhinella marina is common in the cleared tropical forests of the Iquitos region and is regarded as poisonous. The venom from two different populations of R. marina was collected in the Departamento de Loreto, Perú. The samples were assessed for antiproliferative effect and composition. Some 29 compounds were identified or tentatively identified from the venom by spectroscopic and spectrometric means. The main free bufadienolide was marinobufagin 7 while marinobufotoxin 15 and bufalitoxin 9 were the main bufadienolide argininyl diacid derivatives. The alkaloids dehydrobufotenin 28 and bufotenidin 29 were present in both venoms. The main difference in the venoms was the relative ratio of argininyl diacids from bufadienolides to free bufadienolides. The argininyl diacids included derivatives from bufalin, marinobufagin, telocinobufagin, hellebrigenin, resibufogenin and bufotalinin. Four compounds, including undecadienoyl aginine 6 and three argininyl diacids from bufadienolides were tentatively identified for the first time in the samples. The venom showed a strong antiproliferative effect towards MRC-5 normal human lung fibroblasts (0.063-0.247 µg/mL), AGS human gastric adenocarcinoma cells (0.076-0.272 µg/mL), SK-MES-1 human lung cancer cells (0.154-0.296 µg/mL), J82 human bladder carcinoma cells (0.169-0.212 µg/mL), and HL-60 human promyelocytic leukemia (0.071-0.283 µg/mL). The antiproliferative effect is mediated by ROS production and cell cycle arrest in human breast cancer cells (MCF7 and MDA-MB-231). This is the first report on the composition of R. marina venom from the Peruvian Amazon pointing out the need to include different venom samples to get a better picture from the activity and composition of South American toad defense substances.

Biochemical Profile, Biological Activities, and Toxic Effects of Proteins in the Rhinella schneideri Parotoid Gland Secretion.

Parotoid glands of amphibians are known for the production of several biologically active compounds having pharmacological and toxic effects in mammals. In the present work, a protein fraction obtained from Rhinella schneideri parotoid gland (RsPP) was characterized to study its biological and toxic effects. Rhinella schneideri parotoid secretion is composed of up to 30% (w/w) of soluble proteins. Tandem mass spectrometric analysis of the RsPP identified 104 proteins, including actin, beta-actin, ribosomal proteins, catalase, galectin, and uncharacterized proteins; however, no peptidases were found, and this result was reinforced by the absence of proteolytic activity. In addition, RsPP did not exhibit pro-coagulant or antibacterial effects. However, pretreatment of mice with different doses of RsPP intraperitoneally inhibited carrageenan-induced paw edema and increased tissue myeloperoxidase activity. RsPP also reduced interleukin 1ß levels in the peritoneal cavities and cell migration in the peritoneal cavities of an animal model of carrageenan-induced peritonitis. Subchronic treatment of animals with RsPP for 7 consecutive days did not alter the serum biochemical, renal, or liver parameters. However, a significant reduction in blood leukocyte count was observed. Our results showed that R. schneideri parotoid secretion contains proteins with anti-inflammatory and slight toxic effects.

A New Indole Alkaloid from the Toad Venom of Bufo bufo gargarizans.

A new indole alkaloid named bufobutarginine (1), along with three known bufotenines, namely, serotonin (2), bufotenidine (3), and bufotenine (4), were isolated from the water extract of toad venom. Their structures were elucidated by spectral methods. This is the first time that arginine has been found to be involved in the biosynthesis of bufotenines in parotid of toad. The cytotoxic activities of these compounds have been assayed against A375 and A549 cell lines by the MTT method; however, they showed no cytotoxic activities.

Bufadienolides from parotoid gland secretions of Cuban toad Peltophryne fustiger (Bufonidae): Inhibition of human kidney Na(+)/K(+)-ATPase activity.

Parotoid gland secretions of toad species are a vast reservoir of bioactive molecules with a wide range of biological properties. Herein, for the first time, it is described the isolation by preparative reversed-phase HPLC and the structure elucidation by NMR spectroscopy and/or mass spectrometry of nine major bufadienolides from parotoid gland secretions of the Cuban endemic toad Peltophryne fustiger: ψ-bufarenogin, gamabufotalin, bufarenogin, arenobufagin, 3-(N-suberoylargininyl) marinobufagin, bufotalinin, telocinobufagin, marinobufagin and bufalin. In addition, the secretion was analyzed by UPLC-MS/MS which also allowed the identification of azelayl arginine. The effect of arenobufagin, bufalin and ψ-bufarenogin on Na(+)/K(+)-ATPase activity in a human kidney preparation was evaluated. These bufadienolides fully inhibited the Na(+)/K(+)-ATPase in a concentration-dependent manner, although arenobufagin (IC50 = 28.3 nM) and bufalin (IC50 = 28.7 nM) were 100 times more potent than ψ-bufarenogin (IC50 = 3020 nM). These results provided evidence about the importance of the hydroxylation at position C-14 in the bufadienolide skeleton for the inhibitory activity on the Na(+)/K(+)-ATPase.

On frogs, toxins and true friendship: an atypical case report

Abstract The authors report a series of events including the scientific interest for poisonous dendrobates of French Guiana, the human confrontation with the immensity of the evergreen rainforest, the fragility of the best-prepared individuals to a rough life, and the unique and very special manifestation of a solid friendship between two experts and enthusiasts of outdoor life. In the evergreen forest of South America, as in many other scientific field expeditions, everything may suddenly go wrong, and nothing can prepare researchers to accidents that may occur in a succession of uncontrollable errors once the first mistake is done. This is what happened during an expedition in search for dendrobates by an experienced forest guide and naturalist. The authors decided to report the story, considering that it deserved to be brought to the attention of those interested in venomous animals and toxins, in order to illustrate the potential danger of dealing with these organisms.

Toxicity effects of toad (Rhinella jimi Stevaux, 2002) venom in chicken (Gallus gallus domesticus).

This study aimed to evaluate the pathological changes that occur after administering different doses of R. jimi (Stevaux, 2002) parotoid glands secretion to Gallus gallus domesticus chicks. Twenty-three animals were used in this study and were divided into 5 groups that received a toad venom dose of 0, 3.0 mg/kg, 6.0 mg/kg, 10.0 mg/kg, and 25.0 mg/kg. After 48 h, the necropsy and pathological examinations were performed. No clinical signs of toxicity were observed in any group. Macroscopically, hepatomegaly, areas of liver necrosis, splenomegaly, necrotic and hemorrhagic cardiac regions, hydropericardium, dark necrotic lesions of Meckel's diverticulum, and hemorrhages in the lungs and kidneys were detected. Histopathological changes included diffuse vacuolar degeneration of hepatocytes, severe sinusoidal congestion, focal areas of hemorrhage in the parenchyma, swollen cardiac fibers, necrotic myocardial fibers, moderate to acute diffuse alveolar hemorrhage, vacuolar degeneration of the renal tubular epithelium, necrosis of renal tubules, and extensive hemorrhagic areas below the brain and cerebellar meninges. In conclusion, pathological changes of the R. jimi toxins in chicks were noted in the heart, spleen, liver, Meckel's diverticulum, lungs, and kidneys. Most of the changes were similar to those observed in humans and animals exposed to toxins from other toad species.

Poison frog colors are honest signals of toxicity, particularly for bird predators.

Antipredator defenses and warning signals typically evolve in concert. However, the extensive variation across taxa in both these components of predator deterrence and the relationship between them are poorly understood. Here we test whether there is a predictive relationship between visual conspicuousness and toxicity levels across 10 populations of the color-polymorphic strawberry poison frog, Dendrobates pumilio. Using a mouse-based toxicity assay, we find extreme variation in toxicity between frog populations. This variation is significantly positively correlated with frog coloration brightness, a viewer-independent measure of visual conspicuousness (i.e., total reflectance flux). We also examine conspicuousness from the view of three potential predator taxa, as well as conspecific frogs, using taxon-specific visual detection models and three natural background substrates. We find very strong positive relationships between frog toxicity and conspicuousness for bird-specific perceptual models. Weaker but still positive correlations are found for crab and D. pumilio conspecific visual perception, while frog coloration as viewed by snakes is not related to toxicity. These results suggest that poison frog colors can be honest signals of prey unpalatability to predators and that birds in particular may exert selection on aposematic signal design.

Bilirubin attenuates bufadienolide-induced ventricular arrhythmias and cardiac dysfunction in guinea-pigs by reducing elevated intracellular Na(+) levels.

Bufadienolides, known ligands of the sodium pump, have been shown to inhibit the proliferation of several cancer cell types. However, their development to date as anticancer agents has been impaired by a narrow therapeutic margin resulting from their potential to induce cardiotoxicity. In the present study, we examined the effects of bilirubin, an endogenous antioxidant, on the cardiotoxicity of bufadienolides (derived from toad venom) in guinea-pigs. The results showed that bufadienolides (8 mg/kg) caused ventricular arrhythmias, conduction block, cardiac dysfunction and death in guinea-pigs. Pretreatment with bilirubin (75 and 150 mg/kg) significantly prevented bufadienolide-induced premature ventricular complexes, ventricular tachycardia, ventricular fibrillation and death. Bilirubin also markedly improved the inhibition of cardiac contraction in bufadienolide-treated guinea-pigs as evidenced by increases in left ventricular systolic pressure and decreases in left ventricular diastolic pressure in vivo. Furthermore, bilirubin significantly reduced the intracellular sodium content ([Na(+)]( i )) in ex vivo bufadienolide-stimulated guinea-pig ventricular myocytes loaded with the sodium indicator Sodium Green. An antitumor study showed that bilirubin did not compromise the ability of bufadienolides to inhibit gastric cancer cell MGC-803 proliferation. These results suggested that bilirubin can attenuate bufadienolide-induced arrhythmias and cardiac dysfunction in guinea-pigs by reducing elevated [Na(+)]( i ) and may improve bufadienolide therapeutic index in cancer treatment.

Inversely related aposematic traits: reduced conspicuousness evolves with increased toxicity in a polymorphic poison-dart frog.

Prevailing theory contends that aposematic coloration evolves in tandem with toxicity so that the evolution of increased toxicity will accompany the evolution of greater conspicuousness. Although variation in aposematic coloration within single species should be selectively constrained, because individuals varying from a predator-recognized warning signal will incur greater risk of predation, several species of poison-dart frogs display remarkable phenotypic variation. This variation may have evolved to match different levels of toxicity, and these species provide excellent opportunities to examine the evolution of aposematic coloration. Here, I test whether increased conspicuousness in the granular poison-dart frog evolved in tandem with increased toxicity. Contrary to classical predictions, toxicity assays, spectral reflectance measurements, and phylogenetic reconstruction reveal that the less conspicuous color morphs are actually significantly more toxic than the brightest, most conspicuous phenotypes and that the more toxic, less-conspicuous form evolved from a less toxic, more conspicuous ancestor. Through gas chromatography--mass spectrometry analysis of toxin profiles, I traced the increase in toxicity in the less-conspicuous populations to an acquisition of specific alkaloids, some of which are proven convulsants. These results challenge the tenet that increased conspicuousness always evolves with increased toxicity and support the idea that once aposematism has been established in a species, phenotypic variation may evolve from brightness and toxicity becoming decoupled.

Epiquinamide: a poison that wasn't from a frog that was.

In 2003, we reported the isolation, structure elucidation, and pharmacology of epiquinamide (1), a novel alkaloid isolated from an Ecuadoran poison frog, Epipedobates tricolor. Since then, several groups, including ours, have undertaken synthetic efforts to produce this compound, which appeared initially to be a novel, beta2-selective nicotinic acetylcholine receptor agonist. Based on prior chiral GC analysis of synthetic and natural samples, the absolute structure of this alkaloid was established as (1S,9aS)-1-acetamidoquinolizidine. We have synthesized the (1R*,9aS*)-isomer (epi-epiquinamide) using an iminium ion nitroaldol reaction as the key step. We have also synthesized ent-1 semisynthetically from (-)-lupinine. Synthetic epiquinamide is inactive at nicotinic receptors, in accord with recently published reports. We have determined that the activity initially reported is due to cross-contamination from co-occurring epibatidine in the isolated material.

Toad poisoning in three dogs: case reports

Toad poisoning is frequent in dogs, but has been infrequently addressed in published case reports and review articles. Dogs can be poisoned when they bite a toad or otherwise ingest the venom. The venom effects manifest soon after the accident, since the toxin is rapidly absorbed by the mucous membrane of the digestive system. Hospital records of three dogs, diagnosed with toad poisoning, were retrospectively reviewed from January 2005 to July 2007. Poisoned dogs may present only local irritation or systemic signs in the gastrointestinal, cardiac and neurological systems. All three cases presented herein had clinical signs of gastrointestinal alterations including vomiting, sialorrhea and diarrhea. Two dogs developed abnormal cardiac rhythm and two exhibited neurological signs. A poisoned animal requires emergency care and symptomatic therapy with intense monitoring of its clinical parameters. Although there have been reports on the low mortality of dogs poisoned by toads, one animal died even after appropriate therapy. The severity of clinical signs and the risk of death must be considered by the veterinarian.

Intoxicação por veneno de sapo em um canino / Toad venom intoxication in a dog

O sapo do gênero Bufo possui nas suas glândulas paratóides uma secreção mucóide contendo toxinas como bufaginas e Bufotoxinas, que são esteróides cardiogênicos. Os cães podem atacar os sapos, entrando em contato com o veneno por meio das mucosas. Um canino, da raça Bulldog Francês, foi encaminhado ao Setor de Patologia Veterinária da Universidade Federal do Rio Grande do Sul (UFRGS) para a necropsia com histórico de provável intoxicação por veneno de sapo. Na necropsia o canino apresentava pulmões aumentados de volume, avermelhados e com edema, e rins de coloração vermelho-escura. As alterações microscópicas indicaram congestão, hemorragia e edema pulmonar. Nos rins, no baço e nos linfonodos foi observada congestão. As análises toxicológicas para os venenos de rotina foram negativas. Porém, a investigação do veneno de sapo a partir de cromatografia por camada delgada e gasosa demonstrou resultado positivo, revelando ser esta a causa da morte do canino.

The toads of the genus Bufo produce, in their parotoid glands, a mucoid secretion containing toxins such as bufagins and Bufotoxins, which are cardiogenic steroids. The mucous membranes of dogs can absorb this venom when they attack the toads. A French bulldog with a history of probable toad venom intoxication was referred to Veterinary Pathology Section of Federal University of Rio Grande do Sul (UFRGS) for necropsy. The necropsy revealed enlarged, reddish, edematous lungs, and kidneys displaying a dark red color. The microscopic alterations indicated the presence of congestion, hemorrhage, and pulmonary edema. Congestion was observed in the kidneys, spleen and lymph nodes. The routine toxicological analyses for venom detection were negative. Nevertheless, the toad venom test result was positive as assessed by thin layer and gas chromatography, indicating that toad venom intoxication was the cause of death.

Toad poisoning in Laos.

We describe two patients who developed severe illness after eating the skin and eggs of a toad, probably Bufo melanostictus Schneider, in southeastern Laos. One boy died, and one developed a digoxin toxicity-like syndrome with bradycardia and heart failure but survived. A telephone survey of 16 Lao provincial hospitals suggested that toad poisoning occurs in at least six provinces. That 93% of villagers in three villages in southeastern Laos were aware that toads are poisonous but that 51% had encountered patients with toad toxicity suggests that the potential gravity is not appreciated. These data indicate that toad poisoning may be underestimated and that education on the seriousness of toad toxins could be a useful public health measure.

Major biological effects induced by the skin secretion of the tree frog Phyllomedusa hypochondrialis.

Amphibian skin secretions contain several bioactive compounds such as biogenic amines, alkaloids, steroids, proteins and peptides; being peptides a continuously growing field of interest. This work aims to describe the main physiopathological properties of the tree frog Phyllomedusa hypochondrialis skin secretion, obtained by manual stimulation of the dorsal skin surface. Intravenous skin secretion administration provoked lethal effect in mice after 5min. Low doses induced significant systemic and local effects like edema and nociception in mice and topic administration induced myonecrosis in the endothelium of cremaster mice. The presence of phospholipase A(2) activity, proteolytic activity and creatine kinase activity (in the plasma of treated mice) are reported and are very likely to be related to the physiopathological (edematic and myotoxic) activities observed. These data provide in vivo evidence of the complex toxic effects of the P. hypochondrialis skin secretion as well as possible mechanisms of action for these effects.

A common pumiliotoxin from poison frogs exhibits enantioselective toxicity against mosquitoes.

Neotropical poison frogs (Dendrobatidae) contain a variety of lipophilic alkaloids in their diffusely distributed cutaneous glands, including a major class of compounds known as pumiliotoxins. Pumiliotoxins are highly toxic and are believed to protect frogs against predators. Their potential activity against ectoparasites, however, has not been investigated. We tested female yellow fever mosquitoes (Aedes aegypti) for responses to 8-hydroxy-8-methyl-6-(2'-methylhexylidene)-1-azabicyclo[4.3.0]nonane, designated pumiliotoxin 251D [PTX (+)-251D], a skin alkaloid present in all genera of dendrobatids and in other anurans, and to its unnatural enantiomer, PTX (-)-251D. Both enantiomers of PTX 251D presented on silicone feeding membranes reduced landing and feeding by A. aegypti, but PTX (+)-251D did so at lower concentrations. PTX (+)-251D also induced toxicosis, shown when mosquitoes failed to fly off membranes. Similarly, mosquitoes confined with copper wires coated with PTX (+)-251D exhibited greater latencies to fly off the substrate and a higher incidence of leg autotomy than did those confined with the (-)-enantiomer. Our results on the contact toxicities of PTX 251D enantiomers parallel those reported for mice injected with them. The presentation of serial dilutions of PTX (+)-251D to A. aegypti revealed a minimum toxic concentration of 0.1 microg/cm2. This value is substantially lower than that estimated for the cutaneous abundance of this compound in some frogs, an observation consistent the function of PTX 251D in anuran chemical defense against ectoparasitic arthropods.